title: In vitro selection of proteins that undergo covalent labeling with small molecules by thiol-disulfide exchange by using ribosome display.
abstract: There is a great deal of interest in proteins that can bind covalently to target molecules, as they allow unambiguous experiments by tight binding to molecules of interest. Here, we report the generation of proteins that undergo covalent labeling with small molecules through in vitro selection by using ribosome display. Selection was performed from a mutant library of the WW domain with a biotinylated peptide as its binding target, in which the biotin and the peptide are connected by a disulfide bond. After five rounds of selection, we identified mutants carrying a particular cysteine mutation. The binding target reacted specifically with the selected mutant, even in the presence of other proteins, and resulted in the generation of biotin- or peptide-labeled WW domains by thiol-disulfide exchange. When the mutant was fused to a protein of interest, the fusion protein was also labeled with biotin. Thus, the characteristics of the selected mutant should be suitable as a tag sequence that can be covalently labeled with small synthetic molecules. These results indicate that the rapid and efficient generation of such proteins is possible by ribosome display.
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan.
authors.forename: Hayato
authors.initials: H
authors.lastname: Yanagida
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Matsuura
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Kazuta
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Yomo
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001710:Biotin / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D008715:Methionine / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry; D009154:Mutation; D010455:Peptides / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013462:Sulfur Radioisotopes / Q000737:chemistry
chemical_list: D004220:Disulfides; D050505:Mutant Proteins; D010455:Peptides; D013438:Sulfhydryl Compounds; D013462:Sulfur Radioisotopes; D001710:Biotin; D008715:Methionine; D003545:Cysteine